Methods and apparatus for detection of gluten sensitivity, and its differentiation from celiac disease

ABSTRACT

Antibodies are used as biomarkers to assist in distinguishing gluten immune reactivity and sensitivity, silent celiac disease, Crohn&#39;s disease and other gut-related pathologies from classical celiac disease. In one class of embodiments, sera, saliva or other samples from a human or other animal are tested for antibodies to (a) a wheat antigen; (b) a gliadin antigen; and (c) one or more of a wheat germ agglutinin, a gluteomorphin, a glutenin, a deamidated glutenin, a prodynorphin, and a dynorphin. Test results are considered particularly interesting where the wheat antigen and the gliadin antigen are both selected from the group consisting of native and deamidated forms of α-gliadin 33-mer, α-gliadin-17-mer, γ-gliadin-15-mer, ω-gliadin-17-mer, and glutenin 21-mer. Test plates and kits can advantageously test for antibodies to at least three, five, seven or all of mixed wheat antigens, α-gliadin, γ-gliadin, ω-gliadin, glutenin, α-glutenin, wheat germ agglutinin, gluteomorphin, prodynorphins, transglutaminase-2, transglutaminase-3, transglutaminase-6, and gliadin-bound transglutaminase.

The present application is a continuation of U.S. patent applicationSer. No. 15/258,949, filed on Sep. 7, 2016, which is a continuation inpart of U.S. patent application Ser. No. 13/354,119, filed on Jan. 19,2012, which claims the benefit of U.S. Provisional Application No.61/143,4501, filed on Jan. 20, 2011, all of which are incorporated byreference herein in their entirety.

FIELD OF THE INVENTION

The present invention relates to methods and kits for aid in diagnosisof gut-related diseases and pathologies, including at least glutenimmune reactivity and sensitivity, silent celiac disease, and Crohn'sdisease.

BACKGROUND

Wheat allergy, celiac disease and gluten sensitivity are three distinctconditions that are triggered by the ingestion of wheat gliadin (1, 2).These and all other extrinsic materials discussed herein areincorporated by reference in their entirety. Where a definition or useof a term in an incorporated reference is inconsistent or contrary tothe definition of that term provided herein, the definition of that termprovided herein applies and the definition of that term in the referencedoes not apply. In these conditions, the reaction to gluten is mediatedby both cellular and humoral immune responses, resulting in thepresentation of different symptomatologies. For example, in wheatallergy a specific sequence of gliadin peptides cross-links two IgEmolecules on the surface of mast cells and basophils that trigger therelease of mediators such as histamines and leukotrienes (3).

Celiac disease (CD) is an autoimmune condition with known genetic makeupand environmental triggers, such as gliadin peptides. CD affects between1-2% of the general population. Throughout this application, unless thecontext dictates the contrary, all ranges set forth herein should beinterpreted as being inclusive of their endpoints, and open-ended rangesshould be interpreted to include commercially practical values.Similarly, all lists of values should be considered as inclusive ofintermediate values unless the context indicates the contrary.

Markers for confirming a diagnosis of this disorder are IgA againstnative, deamidated gliadin peptides and IgA anti-tissue transglutaminase (tTg) autoantibody. In comparison with 10 CD, glutensensitivity (GS) affects up to 30% of the population (4). According totwo articles published in 2010 and 2011 by Sapone et al (5, 6), symptomsin GS may resemble some of the gastrointestinal symptoms that areassociated with CD or wheat allergy, but it is emphasized that objectivediagnostic tests for gluten sensitivity are currently missing (5, 6).While studying the innate and immune responses in CD compared to thosein GS, the researchers found that TLR1, 15 TLR2 and TLR4, which areassociated with innate immunity, were elevated in mucosal GS but not inCD, while biomarkers of adaptive immunity such as IFN-g, IL-21 andIL-17A were expressed in mucosal tissue in CD but not GS. They believedthat measurements of toll-like receptors and IFN-y, IL-21 and IL-17Awould enable them to differentiate between CD and GS (5, 6) with amethod that is highly invasive and would require a biopsy. Immediatetype 20 hypersensitivity to gluten is IgE mediated, while delayed typehypersensitivity to gluten is an antibody-(IgG, IgA) and T-cell-mediatedreaction, which is called celiac disease or gluten sensitivity withenteropathy (7). In the absence of IgG and IgA against tTg, elevated IgGand IgA against various wheat antigens and peptides indicate the loss ofmucosal immune tolerance against wheat peptides and the development ofgluten sensitivity (7). Due to antigenic similarities between wheatantigens and human tissue, both CD and GS can result in many autoimmuneconditions, including Type 1 diabetes, arthritis, thyroiditis, and evenneuroautoimmune conditions such gluten ataxia and multiple sclerosis(8-10).

It should be appreciated that the term “patients” refer to humans underthe care of a health care professional. More broadly, however, the noveltesting protocols and analyses disclosed herein could be applied tonon-patient humans, and any other animal that could suffer from celiacdisease, gluten sensitivity, and gut-related autoimmunities.

While GS patients, similar to CD patients, are unable to tolerate glutenand can develop the same or similar sets of gastrointestinal symptoms,in GS this immune reaction does not lead to small intestine damage (5,6). This lack of induction of intestinal damage in GS and theassociation of CD with genetic markers HLA DQ2/DQ8 plus small intestinaldamage make the diagnosis of CD much easier than GS. The less severeclinical picture in GS, the absence of tTg autoantibodies, and thedismissal of the significance of elevated IgG and IgA autoantibodiesagainst various wheat proteins and peptides by many clinicians makes GSan extremely dangerous disorder. This is because the persistence of IgGand/or IgA antibodies in the blood for long periods of time, along withinducers of inflammatory cascades can result in full-blown autoimmunity.If this were to be the case, due to the severity of the resulting tissuedamage even implementation of a gluten-free diet might not be able tohelp reverse the course of the autoimmune reaction induced by IgG andIgA antibodies against different wheat antigens and peptides.

A comparison between celiac disease and gluten immunereactivity/sensitivity is shown in FIG. 1. According to this model, iftwo children, one with a negative genetic makeup (HLA DQ2/DQ8−), and theother with positive (HLA DQ2/DQ8+), are exposed to environmentalfactors, such as Rota virus, bacterial endotoxins, and some medicationsor their synergistic effects, the result can be a breakdown of mucosalimmune tolerance in both children. The induction of mucosal immunetolerance against gliadin results in the production of IgA and/or IgGagainst native wheat proteins and peptides, which is the next step inthe initiation of gluten sensitivity in both individuals that are HLADQ2/DQ8− and HLA DQ2/DQ8+.

However, in the individual with the positive genetic makeup, the IgG andIgA antibodies against gliadin along with biomarkers of inflammation canactivate tTg, induce damage to the villi, and result in villous atrophy.Deamidation of a specific gliadin peptide leads to the formation of acomplex between it and the tTg; the presentation of this complex byantigen-presenting cells to T cells and B cells results in IgA or IgGproduction against tTg, deamidated gliadin and the gliadin-tTg complex.The formation of these antibodies and their detection in blood is thehallmark of CD, which is an inherited condition detected in 1-2% of thepopulation. If CD is left untreated, the outcome could be autoimmunitiesand cancer.

In comparison, in an individual negative for HLA DQ2/DQ8, this breakdownin immunological tolerance and the concomitant production of IgA and orIgG against native wheat proteins and peptides may activate aninflammatory cascade. In the absence of tTg activation, however, villousatrophy does not occur. Furthermore, gliadin peptides do not go throughdeamidation, and consequently IgG and IgA antibodies are produced onlyagainst native wheat and gliadin peptides.

With continuous exposure to wheat antigens and continuous mucosal immunetolerance, the wheat antigens and reacting antibodies form an unholyalliance of immune complexes, resulting in severe gluten immunereactivity and sensitivity. This immune reactivity and sensitivity is anon-inherited condition detected in up to 30% of the population. If thisdisorder is left unchecked, prolonged exposure to IgG and IgA antibodiesagainst wheat antigens and peptides and their cross-reaction withdifferent tissue antigens can result in various autoimmune disorders.Therefore, even in the absence of CD, GS might still provide aproductive environment for other gluten-related autoantibodies thatattack different organs.

Furthermore, a gluten-free diet usually is only recommended for thosewho meet the criteria for a diagnosis of CD, not of gluten immunereactivity and sensitivity. Unfortunately, that leaves manygluten-sensitive people suffering unnecessarily with very serioussymptoms that put them at risk for complications, conditions that mightbe resolved with a gluten-free diet, if they only knew.

Thus, a new paradigm is needed for aid in diagnosing and distinguishingamong various gut-related diseases, including gluten immune reactivityand sensitivity, silent celiac disease, celiac disease, and gut-relatedautoimmunity.

SUMMARY OF THE INVENTION

The inventive subject matter of the present invention providesapparatus, systems and methods in which antibodies are used asbiomarkers to assist in diagnosing gluten immune reactivity andsensitivity, silent celiac disease, Crohn's disease and othergut-related pathologies. In some embodiments a mixed wheat antigenpreparation (for example, a mixture of water-soluble and alcohol-solublewheat antigens) can be utilized to differentiate wheat sensitivityand/or wheat-related pathologies (for example, through detection of IgG,IgA, IgM, and/or IgE antibodies that bind to a test surface coated withsuch a mixed wheat antigen preparation) from other pathologies withsimilar symptoms. In such embodiments further differentiation betweenspecific wheat-related pathologies such as celiac disease, gluten immunereactivity and sensitivity, and gluten immune reactivity andautoimmunity being provided by determination of the presence of IgG,IgA, and/or IgM directed to specific (e.g. single molecular species)antigens.

The test results can advantageously be used to assist in differentiatinggluten immune reactivity or sensitivity from celiac disease, especiallywhere the wheat antigen is de-amidated (for example, through the actionof transglutaminase at transglutaminase-sensitive sites or synthesis ofa corresponding peptide sequence), and the gliadin antigen is selectedfrom the group consisting of an α-gliadin-33-mer (SEQ ID NO. 1, SEQ IDNO. 2), an α-gliadin-17-mer (SEQ ID NO.6, SEQ ID NO. 7), aγ-gliadin-15-mer (SEQ ID NO. 10), an ω-gliadin-17-mer (SEQ ID NO. 13,SEQ ID NO. 14), and glutenin 21-mer (SEQ ID NO.15, SEQ ID NO. 16).

In certain aspects of the present invention, whole blood, blood sera,saliva or other samples from a human or other animal are tested forantibodies to one or more of a γ-gliadin protein or a peptide thereof(such as γ-gliadin-15-mer (SEQ ID NO. 10)), an ω-gliadin protein or apeptide thereof (such as ω-gliadin-17-mer (SEQ ID NO. 14, SEQ ID NO.15)) wheat germ agglutinin, a gluteomorphin, a glutenin protein or apeptide thereof (such as glutenin-21-mer), a de-amidated gluteninprotein or a peptide thereof, a prodynorphin, and a dynorphin.

In certain aspects of the present invention, whole blood, blood sera,saliva or other samples from a human or other animal are tested forantibodies to (a) a wheat antigen; (b) a gliadin antigen; and (c) one ormore of a wheat germ agglutinin, a gluteomorphin, a glutenin, ade-amidated glutenin, a prodynorphin, and a dynorphin.

In certain aspects of the present invention, tests are conducted and/ortest results are analyzed for antibodies that assist in distinguishinggluten immune reactivity and/or sensitivity, silent or atypical celiacdisease relative to patently symptomatic (i.e. classical) celiacdisease, Crohn's disease and chronic immune activation. Test plates andkits of particular interest test for antibodies to at least three, five,seven or all of α-gliadin, γ-gliadin, ω-gliadin, glutenin, wheat germagglutinin, gluteomorphin, prodynorphins, transglutaminase, andgliadin-bound transglutaminase (gliadin-trans glutaminase complex). Incertain aspects of the present invention, assays and assay kits ofparticular interest allow for testing IgA and/or IgG antibodies to oneor more wheat antigens, a α-gliadin protein or one or more peptidesthereof such as α-gliadin-33-mer (SEQ ID NO. 1, SEQ ID NO. 2) and/orα-gliadin-17-mer (SEQ ID NO. 6, SEQ ID NO. 7), a γ-gliadin protein orone or more peptides thereof such as γ-gliadin-15-mer (SEQ ID NO. 10),an ω-gliadin protein or one or more peptides thereof such asω-gliadin-17-mer (SEQ ID NO. 13, SEQ ID NO. 14), wheat germ agglutinin,an opioid peptide such as one or more of gluteomorphin, prodynorphinand/or dynorphin, a glutenin protein or one or more peptides thereofsuch as glutenin-21-mer (SEQ ID NO. 15, SEQ ID NO. 16), de-amidatedglutenin protein or one or more peptides thereof, agliadin-transglutaminase complex or peptide derived therefrom (SEQ IDNO. 19, SEQ ID NO. 20), or combinations thereof.

In certain aspects of the present invention, the detection of antibodiescan be performed with an immunoassay, including, but not limited to,ELISA assay, RIA assay, latex agglutination, beads assay, proteomicassays, and other immunoassays known to one of ordinary skill in theart.

Various objects, features, aspects and advantages of the inventivesubject matter of the present invention will become more apparent fromthe following detailed description of preferred embodiments, along withthe accompanying drawing figures in which like numerals represent likecomponents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing differentiation between celiac disease(right hand listing) and gluten immune reactivity/sensitivity (left handlisting), as contemplated herein.

FIG. 2 is a diagram showing the layout of a sample microtiter platehaving 12 different rows with 12 different antigens and peptides.

FIG. 3 is a diagram showing the layout of a sample microtiter plate bywhich IgG or IgA is measured against 12 different antigens or peptidesfrom wheat and associated tissue antigens (antigens or peptides aretransparent).

FIG. 4 is a diagram showing the layout of a sample microtiter plate bywhich IgG or 5 IgA is measured with weekly negative and positivecontrols for quality control purposes (antigens or peptides aretransparent).

FIG. 5 is a diagram of an antibody testing protocol for celiac diseaseusing tTg and various antigens according to the prior art (17).

FIG. 6 is a diagram of a testing protocol for celiac disease usingdeamidated gliadin 10 peptide according to the prior art (17).

FIG. 7 is a diagram of a testing protocol of the inventive concept fordistinguishing wheat-related pathologies from other conditions, andfurther differentiating between wheat-related pathologies (e.g. celiacdisease, gluten immune reactivity and sensitivity, and gluten immunereactivity/sensitivity and autoimmunity) by determining the presence ofspecific antibodies in a sample using a test antigen repertoire thatincludes a mixed wheat antigen preparation, wheat antigens and peptides,and other (e.g. human) according to certain aspects of the presentinvention.

DETAILED DESCRIPTION

According to certain aspects of the present invention, antibodies areused as biomarkers to assist in diagnosing wheat-related pathologies anddistinguishing between gluten immune reactivity and sensitivity, glutenimmune reactivity and sensitivity and autoimmunity, silent celiacdisease, Crohn's disease, and/or other gut-related pathologies asopposed to the classical celiac disease against an array of wheatantigens and peptides.

In certain aspects of the present invention, a bodily fluid is testedfor immunoglobulin G (IgG) and/or immunoglobulin A (IgA) antibodies toone or more of a mixed wheat antigen preparation (i.e. whole-wheatantigen); an α-gliadin protein and/or one or more peptides (at leastsome of which can be deamidated, for example through the action oftransglutaminase or synthesis of a corresponding peptide sequence)thereof; a γ-gliadin protein and/or one or more peptides (at least someof which can be deamidated, for example through the action oftransglutaminase or synthesis of a corresponding peptide sequence)thereof; an ω-gliadin protein and/or one or more peptides (at least someof which can be deamidated, for example through the action oftransglutaminase or synthesis of a corresponding peptide sequence)thereof; a glutenin protein and/or one or more peptides (at least someof which can be deamidated, for example through the action oftransglutaminase or synthesis of a corresponding peptide sequence)thereof; one or more opioid peptides; a gliadin-trans glutaminasecomplex (i.e. gliadin complexed transglutaminase or a correspondingpeptide); transglutaminase; wheat germ agglutinin; human antigens,and/or combinations thereof.

In certain aspects of the present invention, a mixed wheat antigenpreparation (i.e. whole-wheat antigen) can be prepared by combiningwater-soluble and alcohol-soluble proteins extracted from whole wheat,and in some embodiments some or all of such proteins can be deamidated.The α-gliadin protein and/or one or more peptides thereof can includeα-gliadin 33-mer (SEQ ID NO. 1, SEQ ID NO. 2) and/or α-gliadin 17-merSEQ ID NO.6, SEQ ID NO. 7), with other α-gliadin peptides (for example,α-gliadin 25-mer A (SEQ ID NO. 3), α-gliadin 25-mer B (SEQ ID NO. 4),α-gliadin 18-mer (SEQ ID NO. 5), and/or α-gliadin 15-mer (SEQ ID NO. 8,SEQ ID NO. 9) contemplated. The γ-gliadin protein and/or one or morepeptides thereof includes γ-gliadin-15-mer (SEQ ID NO. 10), with otherγ-gliadin peptides (for example, γ-gliadin-20-mer (SEQ ID NO. 11, SEQ IDNO. 12) contemplated. The ω-gliadin protein and/or one or more peptidesthereof includes ω-gliadin 17-mer (SEQ ID NO. 13, SEQ ID NO. 14), withother ω-gliadin peptides contemplated. The glutenin protein or one ormore peptides thereof includes glutenin-21-mer (SEQ ID NO. 15, SEQ IDNO. 16), with other glutenin peptides contemplated. It should beappreciated that such gliadin proteins and peptides can, in someembodiments of the inventive concept, be deamidated. Such deamidationcan be the result of the action of transglutaminase, which providesdeamidation of specific glutamine residues within the protein/peptidesequence, or can be the result of peptide synthesis that replicates theamino acid sequence produced by the action of transglutaminase on acorresponding native peptide sequence. It should be appreciated that thesites of transglutaminase activity are known, and as such the amino acidsequence of such deamidated species is either known or can be readilyderived. It should also be appreciated that deamidated peptides can bederived from transglutaminase treatment of native sequences or bysynthesis of peptides having sequences corresponding to native sequenceshaving been subjected to transglutaminase activity.

In some embodiments of the inventive concept one or more opioid peptidescan be used to determine the presence of antibodies to such peptides,with such characterization providing useful differentiation betweenwheat-related pathologies. Such one or more opioid peptides includesexorphin peptides including gluteomorphin, prodynorphin and/ordynorphin, with other exorphin peptides contemplated. In someembodiments of the inventive concept one or more human antigens can beused to determine the presence of antibodies to such human antigens,with such characterization providing useful determination betweenwheat-related pathologies. Such human antigens can be proteins orpeptides. Suitable human antigens include glutamic acid decarboxylase(GAD), a cerebellar peptide (i.e. a peptide derived from a cerebellarprotein), and other human tissue antigens.

In certain aspects of the present invention, whole blood, bloodserum/sera, saliva or other bodily fluid samples from a human or otheranimal are tested for antibodies to (a) a mixed wheat antigenpreparation (i.e. whole wheat antigen); (b) a gliadin antigen; and (c)one or more of a wheat germ agglutinin, gluteomorphin, a glutenin orglutenin peptide (e.g. an α-, γ-, and/or ω-glutenin or gluteninpeptide), a de-amidated glutenin or glutenin peptide (e.g. an α-, γ-,and/or ω-glutenin or glutenin peptide deamidated through the activity oftransglutaminase or having a corresponding sequence), a prodynorphin,and a dynorphin. Test results are considered particularly interestingwhere a wheat-derived peptide antigen is deamidated, and the gliadinantigen is selected from the group consisting of α-gliadin 33-mer (SEQID NO. 1, SEQ ID NO. 2), α-gliadin 17-mer (SEQ ID NO. 6, SEQ ID NO. 7),α-gliadin 15-mer (SEQ ID NO. 8, SEQ ID NO. 9), ω-gliadin 17-mer (SEQ IDNO. 13, SEQ ID NO. 14), and glutenin-21-mer (SEQ ID NO. 15, SEQ ID NO.16). Test plates and kits can advantageously test for antigens to atleast three, five, seven or all of α-gliadin (and/or peptides thereof),γ-gliadin (and/or peptides thereof), ω-gliadin (and/or peptidesthereof), glutenin (and/or peptides thereof), wheat germ agglutinin,gluteomorphin, one or more prodynorphins, transglutaminase, andgliadin-bound transglutaminase (i.e. gliadin-transglutaminase complex).In certain aspects of the present invention, assays and assay kits ofparticular interest allow for testing IgA and/or IgG antibodies to oneor more of mixed wheat antigens, α-gliadin protein or one or morepeptides thereof such as α-gliadin-33-mer (SEQ ID NO. 1, SEQ ID NO. 2)and/or α-gliadin-17-mer (SEQ ID NO. 6, SEQ ID NO. 7), γ-gliadin proteinor one or more peptides thereof such as γ-gliadin-15-mer (SEQ ID NO.10), ω-gliadin protein and/or one or more peptides thereof such asω-gliadin-17-mer (SEQ ID NO. 13, SEQ ID NO. 14), wheat germ agglutinin,an opioid peptide such as one or more of gluteomorphin, prodynorphinand/or dynorphin, glutenin or one or more peptides thereof such asglutenin 21-mer (SEQ ID NO. 15, SEQ ID NO. 16), de-amidated gluteninprotein or one or more peptides thereof, gliadin-transglutaminasecomplex or corresponding peptide (SEQ ID NO. 19, SEQ ID NO. 20), orcombinations thereof.

In certain aspects of the present invention, the detection of antibodiescan be performed with an immunoassay, including, but not limited to,ELISA assay, RIA assay, latex agglutination, beads assay, proteomicassays, and other immunoassays known to one of ordinary skill in theart.

Following are exemplary descriptions of assays, and their use andanalysis with respect to some test patients. Although other materialsand methods similar or equivalent to those described herein can be usedin the practice or testing of the present invention, the preferredmethod and materials are now described in the exemplary description ofassays to further illustrate the present invention.

Example 1

ELISA Assay

A. Materials and Methods—Plate and Sample Preparation:

Wheat antigens and peptides. A mixed wheat antigen preparation (i.e.whole-wheat antigen) was prepared by combining water-soluble andalcohol-soluble proteins. Different peptides included glutenin 21-mer(SEQ ID NO. 15, SEQ ID NO. 16) and gliadin peptides including α-gliadin33-mer (SEQ ID NO. 1, SEQ ID NO. 2), α-gliadin 17-mer (SEQ ID NO. 6, SEQID NO. 7), γ-gliadin 15-mer (SEQ ID NO. 10), and ω-gliadin 17-mer (SEQID NO. 13, SEQ ID NO. 14). In some embodiments deamidated peptidescorresponding to one or more of these sequences were produced bysynthesizing a peptide sequence corresponding to the sequence of thecorresponding native peptide when subjected to transglutaminaseactivity, where the resulting synthetic peptide reflected a sequencedeamidated at transglutaminase-susceptible sites. For example, in someinstances a deamidated glutenin 21-mer peptide (SEQ ID NO. 16) wasprovided by synthesis of a peptide corresponding to that of thecorresponding glutenin 21-mer following selective deamidation bytransglutaminase. Peptides were synthesized by Bio-Synthesis Inc.(Lewisville, Tex.). Gluteomorphin, prodynorphin, transglutaminases,gliadin-transglutaminase complex (i.e. gliadin bound totransglutaminase), and glutamic acid decarboxylase (GAD-65) were of HPLCgrade. Wheat germ agglutinin (WGA) was purchased from Sigma/Aldrich(Saint Louis, Mo.).

The antigens and peptides were dissolved in methanol at a concentrationof 1.0 mg/mL, then diluted 1:100 in 0.1 M carbonate-bicarbonate buffer,pH 9.5, and 50 μL were added to each well of a polystyrene flat-bottomELISA plate. The ELISA plates were incubated overnight at 4° C. and thenwashed three times with 200 μL Tris-buffered saline (TBS) containing0.05% Tween 20 (pH 7.4). Non-specific binding of immunoglobulins fromsamples being characterized was prevented by adding 200 μL of 2% bovineserum albumin (BSA) in TBS to the wells and incubated overnight at 4° C.ELISA plates were washed and after conducting quality control were keptat 4° C. until used.

The enzyme conjugates included: Affinity Purified AntibodyPhosphatase-labeled Goat anti-Human IgG (Jackson ImmunoResearch, Cat#109-055-008), and Affinity Purified Phosphatase-labeled Goat anti-HumanIgA Antibody (Jackson ImmunoResearch, Cat #109-055-011).

Other additional reagents and materials included in the method asfurther described herein, include: Phosphate-Buffered Saline Powder(Sigma, Cat # P3813-10PAK), Bovine Serum (Sigma, Cat # P1379-1000ML),Glycerol (Sigma, Cat # GSSI6-S00ML), Sodium Hydroxide (Sigma, Cat #S-S881), Magnesium Chloride (Sigma, Cat #8266), Diethanolamine (Sigma,Cat # D-888S), 1.0 N Hydrochloric Acid Solution (Sigma, Cat # H3162), 5mg Substrate Tablets: p-NPP (para-nitrophenyl phosphate) (Sigma, Cat #S-0942), and Distilled water (D. H20).

The microwell plates were prepared and coated with the desired numberand types of wheat- and/or human-associated antigens and/or peptides. Inthe following case examples, 12 different wheat associated antigens andpeptides were coated on the microwell plates. Calibrators, positivecontrols, and diluted patient samples were added to the wells andantibodies recognizing the antigens coated within the test wells wereallowed bind during the first incubation. After washing the wells toremove unbound proteins, purified alkaline phosphatase labeled rabbit orgoat anti-human IgG and/or anti-human IgA was added to the wells andincubated in a second incubation step. Following the second incubationstep unbound alkaline phosphatase labeled antibodies were removed by afurther wash step.

Bound alkaline phosphatase (indicating the presence of an IgG:boundantigen or IgA:bound antigen complex within a test well) was visualizedby adding paranitrophenyl phosphate (PNPP) substrate, which gives ayellow reaction product, the intensity of which is proportional to theconcentration of IgG or IgA antibody specific for the antigen coatedwithin the test well in the sample. Sodium hydroxide was added to eachwell to stop the reaction. The intensity of color (i.e. optical density)was read at 405 nm.

Plain red top or red “tiger top” tubes (SST tubes) were used forspecimen collection, although in certain aspects, other specimencollection apparatus are contemplated for this assay. For example, useof heparin or EDTA plasma is also contemplated. Blood samples werecollected using aseptic venipuncture techniques and serum was obtainedusing standard procedures. In certain aspects it is preferred that aminimum of about 500 μL of serum for the assay, which thereforecorresponds to about 0.5 mL or more of blood.

B. Test Assay Procedure

The analytical procedure for IgG and/or IgA antibody array to assist indiagnosing and detection of gluten immune reactivity and sensitivity,silent celiac disease, Crohn's disease and other gut-related pathologiesis now discussed. In particular, such assays can be used todifferentiate wheat-related pathologies from other pathologies havingsimilar symptoms and to differentiate or assist in differentiatingbetween celiac disease, gluten immune reactivity and sensitivity, andgluten immune reactivity and reactivity and autoimmunity. In someaspects, all reagents were allowed to reach room temperature before thetest assay was commenced. The test assay procedure includes preparingthe desired number of coated wells or plates with the desired number andtype of wheat-associated antigens and/or peptides. Once the microtiterwells are prepared, about 100 μL of 1:100 diluted control or calibratorare added to Rows A and B of the microtiter plate as shown in FIG. 3using a multi-channel pipettor, incubated, and nonspecific bindingblocked as described above. About 100 μL of 1:100 diluted patient's testsample, for example blood serum, was added to duplicate wells of rows Cand D for Clinical Specimen 1, rows E and F for Clinical Specimen 2, androws G and H for Clinical Specimen 3. On a separate plate, the periodicnegative and positive controls similar to clinical specimens induplicates were conducted, as shown in FIG. 4. The plates were thenincubated for 60 minutes at room temperature. After incubation, thewells were emptied and washed four times with PBS using an ELISA Washer.About 100 μL of optimally diluted alkaline phosphatase-labeled goatanti-human IgA was added to the IgA plate or about 100 μL ofenzyme-labeled anti-human IgG was added to the IgG plate at optimaldilution.

The respective plates were then incubated for 30-60 minutes at roomtemperature. About ten minutes before the conjugate-incubation ended, asubstrate solution was prepared by mixing 5 mg of a p-nitrophenylphosphate tablet with 5 ml of substrate buffer, which was mixed welluntil the tablet completely dissolved. Following incubation with enzymelabeled anti-human IgA or IgG the plates were washed four times with PBSusing an ELISA plate washer. Then, about 100 μL of substrate solutionwas added to each well. The plate was then incubated for 30 minutes atroom temperature with the avoidance of any exposure to direct sunlight.The reaction was stopped by adding about 50 μL of 3 N NaOH. The colorintensity (i.e. optical density) of the wells at 405 nm were read usinga microtiter plate reader against a blank well, with the absorbancevalues of the calibrators, controls and unknown samples being recorded.

C. Calculation of Results

After the plate was read the plate at 405 nm to obtain the opticaldensity values (OD405), the mean ODs of the negative controls, the meanODs of the positive controls and the mean ODs of each clinical specimenwere divided by the mean ODs of calibrators on Rows A and B to obtaineach Index Value (IV). The Index Value (IV) for each antibody wascalculated against the 12 different antigens by dividing the mean OD ofeach duplicate sample by the mean OD of the calibrator control value(for example, divide the mean OD of wells C1 and D1 by the mean OD ofwells A1 and B1, the mean OD of wells C2 and D2 by the mean OD of wellsA2 and B2, the mean OD of wells C3 and D3 by the mean OD of wells A3 andB3, etc.). The results were then compared to the established referenceranges. A sample calculation is shown below:

Index Calculation for Wheat Antigens:

Index=(Mean OD of Patients)÷(Mean OD of Calibrators)

Cal 1 (OD) 0.41 Cal 2 (OD) 0.44 Sample 3 A (OD) 3.77 Sample 3 B (OD)3.79 Index 8.93

D. Interpretation of Results

Examples of IgG and IgA antibody patterns of 3 patients with celiacdisease, 3 patients with gluten immune reactivity and sensitivity, and 3patients with Crohn's disease with overlapping gluten sensitivity areshown in Tables 2-7. Crohn's disease, also known as regional enteritis,is a type of inflammatory bowel disease having a strong geneticcomponent. It is often described as an autoimmune disease, but othersconsider it be a disease of immune deficiency.

Data interpretation and laboratory differentiation between celiacdisease and gluten immune reactivity/sensitivity/autoimmunity aresummarized in Table 8. Peptide sequences for peptides designated inTable 8 and other peptides that are contemplated as useful in suchdifferentiation can be found in Table 9. It should be appreciated thatthe sequences indicated for gliadin-transglutaminase complex A andgliadin-transglutaminase complex B in Table 9 show the relevant gliadinpeptide sequence in parenthesis and imbedded within the relevanttransglutaminase peptide sequence, with the relevant gliadin peptidesequence positioned immediately to the right of transglutaminase lysine(K) residue to which it is complexed. In particular, it is nowcontemplated that celiac disease can be differentiated from silentceliac disease, gluten immune reactivity/sensitivity and/orgluten-related autoimmunity as follows:

a. A serological pattern of silent celiac disease is indicated where thetest results include positive results of IgA and/or IgG against any orcombination of mixed wheat proteins (i.e. whole wheat antigen),α-gliadin 33-mer (SEQ ID NO. 1), deamidated α-gliadin 33-mer (SEQ ID NO.2), α-gliadin 25-mer (SEQ ID NO. 3, SEQ ID NO. 4), α-gliadin 18-mer (SEQID NO. 5), α-gliadin 17-mer (SEQ ID NO. 6), γ-gliadin 15-mer(SEQ ID NO.10), ω-gliadin 17-mer (SEQ ID NO. 13, SEQ ID NO. 14), glutenin,deamidated glutenin, gluteomorphin, prodynorphin, and wheat germagglutinin, and negative results against tranglutaminase-2, and at leastone of IgA and IgG tests positive against at least one oftransglutaminase-3 and transglutaminase-6.

b. A serological pattern of gluten immune reactivity and sensitivity isindicated where the test results include positive results for IgG and/orIgA against mixed wheat proteins (i.e. whole wheat antigen), nativeα-gliadin, γ-gliadin, ω-gliadin, glutenin, deamidated glutenin,gluteomorphin, and wheat germ agglutinin, but not deamidated α-gliadinand not transglutaminase-2.

c. A diagnosis related to gluten immune reactivity/sensitivity withautoimmunity is indicated where the test results include a positive forIgG and/or IgA against mixed wheat proteins (i.e. whole wheat antigen),native α-gliadin and/or peptides thereof, γ-gliadin and/or peptidesthereof, ω-gliadin and/or peptides thereof, glutenin and/or peptidesthereof, deamidated glutenin and/or peptides thereof, gluteomorphin, andwheat germ agglutinin, and positive results for any IgG, IgA or IgM tohuman antigens such as glutamic acid decarboxylase, cerebellar tissueantigens or peptides and/or other human tissue antigens, and negativeresults for IgA to deamidated α-gliadin, deamidated glutenin or againsttransglutaminase-2, but positive IgA or IgG against transglutaminase-3and/or transglutaminase-6.

Reportable ranges and reference ranges are show in Table 1, which may beupdated from time to time. The reader will note that compared toestablished reference ranges at 2 standard deviations above the mean,while IgA antibody against tTg and gliadin-tTg complex is highlypositive (confirming the diagnosis of CD), the pattern and the strengthof IgG and IgA antibody varies from antigen to antigen. For example,while both IgG and IgA against wheat antigens in all three patients withceliac disease was 4-6 fold higher than reference ranges, when the IgGand IgA was measured against α-gliadin 33-mer the IgG antibody level wassignificantly elevated in one and the IgA level in two out of threepatients (Tables 2 and 3).

When compared to patients with celiac disease, in patients with glutenimmune reactivity and sensitivity none of the three patients showed asignificant IgA reactivity against tTg and gliadin-tTg complex, whilethe IgG and IgA antibodies against mixed wheat proteins and incombination with one or more wheat peptides (α-gliadin and/or peptidesthereof, γ-gliadin and/or peptides thereof, ω-gliadin and/or peptidesthereof, glutenin and/or peptides thereof, gluteomorphin, prodynorphinand wheat germ agglutinin) was significantly elevated. The IgA immunereaction against mixed wheat proteins and wheat derived peptides incombination with tTg and gliadin-tTg complex clearly distinguishedbetween celiac disease and gluten immune reactivity/sensitivity, inwhich IgG and/or IgA antibodies are reactive against various wheatantigens and peptides but not against tTg and the gliadin-tTg complex(Tables 4, 5).

Tables 6 and 7 present the results of three patients with Crohn'sdisease. The pattern of IgG and IgA antibodies against tTg andgliadin-tTg complex clearly shows that these patients, in addition toCrohn's disease, are also suffering from gluten immunereactivity/sensitivity, and possibly also celiac disease (Tables 6, 7).

CASE STUDY EXAMPLES

Four different case reports, the first on a patient with celiac disease,the second with 10 gluten sensitivity, the third with gluten sensitivityand autoimmunity, and the fourth with gluten sensitivity overlappingwith Crohn's disease are shown below.

A. Case Report #1: Diagnosis of Celiac Disease in the Elderly by the Useof IgA against Gliadin and Tissue Transglutaminase with Improvement on aGluten-Free Diet

A 76 year-old man with longstanding dyspepsia, indigestion, tirednessand rapid weight loss was referred for gastrointestinal evaluation.Blood tests showed macrocytic anemia with low concentrations of folateand vitamin B-12. The patient's hemoglobin concentration was 79 g/L,albumin 32 g/L, and transglutaminase 212 μg/mL (normal range=0-10 μg/mL.An urgent colonoscopy and duodenal biopsy was performed, which yieldedmacrocospically normal results. At this level his IgG and IgAconcentrations against gliadin and transglutaminase were checked usingFDA-approved kits. Both IgG and IgA against α-gliadin were very high;against transglutaminase, IgA but not IgG was 3.8-fold higher than thereference range. In view of the IgA positivity against gliadin andtransglutaminase and diagnosis of celiac disease he was transfused with2 units of packed cells and started on both a gluten-free diet and 20 mgof prednisone daily. Six months later he had gained about 12 pounds andshowed few GI symptoms. Because of this improvement the patient becamecommitted to the gluten-free diet. One year after the first performanceof IgG and IgA antibody testing against gliadin and transglutaminase therepeat tests for these antibodies were negative, which is a furtherindication that disease management plus a gluten-free diet wasinstrumental in the treatment of this elderly patient with a silentceliac disease.

Discussion: According to Catassi et al. (1, 11), celiac disease (CD) isone of the most common lifelong disorders in western countries. However,most cases of CD remain 10 undiagnosed mostly due to the poor awarenessof the primary care physician regarding this important affliction(Catassi C, et al., Am J Gastroenterol, 102:1454-1460, 2007). Celiacdisease is perceived as presenting GI symptoms accompanied bymalabsorption. But many patients with celiac disease do not present GIsymptoms. These individuals may have silent or atypical celiac disease,and the condition may present with iron deficiency, anemia, increasedliver enzymes, osteoporosis or neurological symptoms (12). As usedherein, the term “atypical celiac disease” refers to celiac disease inpatients who have only subtle symptoms, and the term “silent celiacdisease” refers to celiac disease in patients who are asymptomatic.

The increasing recognition of celiac disease is attributed to the use ofnew serological assays with higher sensitivity and specificity. Untilrecently celiac disease was incorrectly 20 perceived as being uncommonand detected mainly during infancy or childhood. However, it is nowrecognized that most cases of CD occur in adults 40-60 years old.Patients in this age group may present their symptoms, lab test resultsand other examination signs in atypical fashion. In fact, according to avery recent publication, less than one in seven patients is correctlydiagnosed with CD (13). Consequently, as this case shows, if an adultpatient presents with symptoms and signs suggesting malabsorption,testing for IgA antibody against gliadin and trans glutaminase should beconsidered. If the test results are positive, celiac diseases shouldthen be made a part of the differential diagnosis, based on which agluten-free diet should be recommended. If the gluten-free diet shouldproduce an improvement in symptoms, the patient should commit to thediet regardless of age.

B. Case Report #2: Gluten Immune Reactivity and Sensitivity induced by aCombination of Anesthetics, Antibiotics and Pain Medication

A 46-year-old woman was given her yearly checkup by her internist. Basedon her medical examination and a normal CBC, chemistry including liverenzymes, and an autoimmune profile, she was classified as a healthyperson. Gluten antibodies were not measured at that time. A few monthslater she went to her dentist for a root canal, bone graft andpreparation for dental implantation. During five different visits overten days she was treated with anesthetic material (mepivacaine),antibiotics and painkillers. Four months later the dental implantprocedure was completed after local anesthesia with lidocaine withsubsequent prescription of antibiotic (amoxicillin) and painkillers.Four hours later she developed a severe allergic reaction with localizededema, in particular the lips and periorbital area swelling. The patientbecame agitated and exhibited with a generalized itching, particularlyher face, hands and feet. Tightness of the chest with wheezing anddifficulty in breathing was an indication of allergic reaction to one ormore of the medicines used. She was immediately treated with 0.01 mL perkg of body weight of adrenaline, intramuscularly supplemented byantihistamine treatment. However, while the allergic reaction wascontrolled, the patient developed severe vomiting and diarrhea withsevere abdominal pain, which lasted for 8 days. Two weeks later, whilethe diarrhea had ameliorated, the patient continued to complain aboutbloating and abdominal discomfort with irritable bowel¬like syndrome.She was referred to a GI specialist who detected nothing of note upon athorough examination. The possibility of gluten sensitivity was thenconsidered, and the patient was tested for HLA typing and IgG and IgAanti-gliadin and anti-transglutaminase antibodies. Immunological testsshowed these results: IgG anti-gliadin 6.8 U/mL (normal range <20 U/mL);IgA anti-gliadin 4.1 U/mL (normal range <20 U/mL); IgG anti-tTg 2.1 U/mL(normal range <6 U/mL); IgA anti-tTg 1.2 U/mL (normal range <4 U/mL);and negative for HLA DQ2 and DQ8. Based on these findings glutensensitivity and celiac disease were excluded, and it was concluded thatthe patient was suffering from psychogenic or idiosyncratic reactionassociated with reaction to the anesthetic and its synergistic effectwith the antibiotics. Ninety days later upon her follow-up visit thepatient was still complaining about bloating and abdominal pain,particularly 1-3 hours after each meal. Repeat testing was ordered, andboth a basic test and a comprehensive test was ordered for anti-gliadinand -tTg IgG and IgA along with ASCA and p¬ANCA IgG, which are thesuggested tests for suspected Crohn's disease and ulcerative colitis.Interestingly, almost 100 days after the first GI discomfort, while ASCAand p-ANCA were completely within the normal range, both the IgG and IgAagainst gliadin were 4 to 9-fold above the reference range (gliadinIgG=79 U/mL; IgA=54 U/mL). However, IgG and IgA antibodies against tTgwere within the normal range. In addition, the IgG and IgA antibodytesting was performed against an array of wheat, gliadin, glutenin,wheat germ agglutinin, gliadin-tTg complex and tTg antigens. IgG wasdetected against 8 out of 12 tested antigens and IgA against 6 out of 12tested antigens at 2-7 fold higher than established reference ranges.Both IgG and IgA against tTg and gliadin-tTg complex were negative (seeTables 4 and 5, Sample #1). These results along with the positivity ofthe basic IgG and IgA test against gliadin but not againsttransglutaminase showed that the patient, due to allergic reaction toenvironmental factors, had lost tolerance to wheat antigens and haddeveloped gluten sensitivity but not celiac disease. Despite the absenceof elevation in IgG and IgA levels against tTg, due to the continuous GIdiscomfort and the elevated IgG and IgA against gliadin, a gluten-freediet was recommended, and a dietitian advised the patient to takeprobiotics and go on a restricted diet free of glutens and also oflectins, since the WGA level was also elevated for both IgG and IgA. Sixmonths after the introduction of the diet and the probiotics, thepatient's GI discomfort had subsided and she was back to normal health.

Discussion: The term gluten sensitivity refers to a state of heightenedimmunological responsiveness to gluten as indicated by the elevation ofIgG, IgA or both against gliadin but not against transglutaminase (14).Gluten sensitivity begins with the loss of mucosal immune tolerance towheat antigens and peptides due to environmental factors affecting themucosal immune homeostasis.

In this case gluten sensitivity was confirmed based on GI symptoms andimmunological testing, in particular IgG and IgA against gliadin and itsassociated proteins and peptides almost 100 days after the triggeringfactors had affected her state of immunological tolerance to wheat andassociated antigens. It seems that in this patient the synergisticeffects of anesthetics, antibiotics and painkillers resulted indysregulation of her mucosal immune system, followed by a breakdown inimmunological tolerance to wheat and other dietary proteins andpeptides. This, possibly in combination with the effect of environmentalfactors on the activity of the digestive enzymes, resulted in theinduction of the opening of tight junctions and the entry of undigestedwheat proteins and peptides into the submucosa, lymph nodes, and thecirculation. These antigens were subsequently presented byantigen-presenting cells to T cells and B cells. During this processgliadin-specific B cells are assisted by gliadin-specific T cells,leading to B-cell clonal expansion and the release of IgG and IgAantibodies to gliadin and associated proteins and peptides, which inthis case was detected about 100 days after the original traumaticexperience.

It is concluded herein that screening for gluten sensitivity in patientswith GI discomfort associated with the use of anesthetics andantibiotics may be easily and cost-effectively undertaken by measuringcirculating IgG and IgA against gliadin and associated proteins andpeptides. Failure to do so may not only deprive the patient of anaccurate diagnosis and the proper treatment by implementation of agluten-free diet, but may also result in unnecessary medicalinterventions with their associated side effects.

C. Case Report #3: Gluten Immune Reactivity, Sensitivity andAutoimmunity

Here, a case report is described in which the original presentation ledto an erroneous diagnosis of irritable bowel syndrome, resulting inincorrect medical intervention. The correct diagnosis of gluten immunereactivity and sensitivity was made after years of mistreatment.

A 49-year-old woman with abdominal pain, constipation, acid reflux andheadache was examined by an internist. Investigation revealed normal CBCwith hemoglobin of 10.8 g/dl and normal chemistry profile includingliver enzyme. Over several visits detailed biochemical and immunologicalprofiles including ANA, rheumatoid factor, T3, T4, and TSH levels wereperformed, all testing within the normal range. After repeatedcomplaints about GI discomfort, the patient was referred for GIevaluation. Both endoscopy and H. pylori test results were normal. Thepatient was diagnosed with irritable bowel syndrome and put on—blockersand nexium, which moderately improved her symptomatologies. Four yearslater, however, in addition to the old GI symptoms and headache, shepresented symptoms of malaise, blurred vision and facial rash. She wasintermittently sleepy and irritable, and experienced breathing problems.Further lab tests revealed her hemoglobin was 9.7 g/dl with MCV of 72fL, a raised erythrocyte sedimentation rate (46 mm/1st hour), ANA of1:80 (normal range <40), mild elevation in IgA smooth muscle antibody,double-stranded DNA and extractable nuclear 10 antibodies were negative.Based on the available evidence, a diagnosis of systemic lupuserythematosus (SLE) was made by a rheumatologist, and treatment withsteroids was commenced. There was some improvement in her overall statebut her hemoglobin level continued to be low, while her ESR fluctuated.Two years later she developed difficulty in passing urine accompanied bytingling and sensory disturbance in her trunk and legs, which led to herbeing referred to a neurologist. Close questioning revealed a band-likesensation in the trunk and reduced visual acuity (8/46 in the right eye,8/23 in the left eye) with minimal eye pain, but normal eye movement.Lab investigation came up with low hemoglobin, abnormal MCV, and lowserum ferritin at 14 μg/L (normal range 10-150 μg/L), which confirmediron deficiency. MRI scan of the brain showed extensive white matterabnormalities not typical of multiple sclerosis, but no abnormalitieswere detected in CSF examination. While blood and CSF examination showedno evidence of bacterial and viral infection including syphilis,mycobacteria, borrelia, EBV, CMV, HTLV, and Herpes Type-6, visual evokedpotentials showed delay in both optic nerves. In view of theseabnormalities, and since tests for gluten sensitivity had not beenperformed during the earlier investigations, the possibility of glutensensitivity was considered. A comprehensive IgG and IgA panel wasordered against a repertoire of wheat proteins and peptides, as well asagainst tTg and various tissue antigens. This comprehensive glutensensitivity and immune reactivity screen revealed IgG against wheatantigens, α-gliadin 33- and 17-mer, γ- and ω-gliadin, glutenin,gluteomorphin, prodynorphin, gliadin-tTg complex, wheat germ agglutinin,and glutamic acid decarboxylase 65 (GAD-65). IgA antibodies weredetected against wheat antigens and wheat germ agglutinin (see Tables 4and 5, Sample #3). Interestingly, both IgA and IgG tested against tTgwere within the normal range. Furthermore, antibodies againstganglioside, cerebellar, synapsin, myelin basic protein, collagen,thyroglobulin and thyroid peroxidase were tested, and all were 2-4 foldabove the reference range. Upper GI endoscopy and biopsy revealed normalhistology and intraepithelial lymphocytes. Overall the patient wasdiagnosed as having gluten sensitivity with its associatedautoimmunities, including gluten ataxia, headache, white matterabnormalities, and neuromyelitis optica. A five-day course ofintravenous methylprednisolone was implemented, and gradually thesensory, motor and visual symptoms improved. In addition, based on thevery high levels of IgG and some IgA antibodies against a repertoire ofwheat antigens and peptides, a gluten-free diet was introduced, and 12weeks later marked improvement was observed in the patient's clinicalsymptomatology. She continued the 100% gluten-free diet under theobservation of a dietitian, and the steroid treatment was stopped. Sixmonths after introduction of the diet antibody tests against wheatantigens, peptides, and human tissue were repeated; more than 60%reduction in antibody levels was observed, and the patient became almostasymptomatic. Discussion: From this data it was concluded that a patientmay suffer from gluten immune reactivity and sensitivity without havingabnormal tissue histology or flat erosive gastritis and antibody againsttTg based on which a diagnosis of celiac disease is normally made. Ifpatients with gluten sensitivity and immune reactivity are not detectedin time based on the proper lab tests, in particular IgG and IgAantibodies against a repertoire of wheat proteins and peptides,patients' symptomatologies may mislead many clinicians into treatingtheir patients for lupus, MS-like syndrome, neuromyeltitis optica, andmany other autoimmune disorders.

Therefore, measurement of IgG and IgA antibodies against a repertoire ofwheat antigens and peptides is recommended for patients with signs andsymptoms of autoimmunities so that intervention with a gluten-free dietwill be instrumental in reversing the autoimmune conditions associatedwith gluten immune reactivity and sensitivity. Otherwise, untreatedand/or mistreated, the patient will develop multiple autoimmunedisorders.

D. Case Report #4: Gluten Immune Reactivity and Sensitivity Overlappingwith Crohn's Disease

Crohn's disease is an inflammatory disorder that often emerges duringthe second or third 20 decade of life, affecting the terminal ileum inmore than two-thirds of patients (15). A combination of genetic andenvironmental factors, including a shift in gut flora and dysfunctionalresponses against them, is believed to lead to dysregulated immunity,altered intestinal barrier function, and possibly autoimmunity (16).

Here, a 32-year-old man presented with gastrointestinal discomfort anddiarrhea 2-3 times per month. Laboratory results including chemistrypanel, CBC, iron, ferritin, transferrin, vitamin B-12, thyroid function,and urine analysis were within the median level of the normal range.Upon the second visit and continuation of GI symptoms he was referred toa GI specialist who ordered additional lab examinations. These testswere microbiological evaluation of the stool and blood tests forantibodies against H. pylori, Saccharomyces and gliadin. Stool testingwith respect to the detection of Salmonella, Shigella, Yersinia,Campylobacter, enteropathogenic and enterohemorrhagic E. coli orClostridium difficile came out negative. Regarding antibody examinationsin the blood, IgG against H. pylori and IgA against Saccharomyces andgliadin were negative, but IgG against gliadin was moderately elevatedat 59 U/mL (normal values=<20 U/mL). The IgG antibody elevations wereconsidered non-specific or protective, and the patient was put onpainkillers and sent home with no diagnosis of any specific disorder.Three years later after seeing the frequency of the watery diarrheaincrease to 3-5 times daily and losing 12 pounds of his body weight inthe last two months, the patient went to another GI specialist for asecond opinion. Gastric and duodenal biopsies and endoscopy wereperformed. While the endoscopy of the upper GI tract revealed gastritisof the antrum, histologically, gastric and duodenal biopsy turned out tobe negative. D-xylose absorption test was performed; the resulting valueof 1.89 g/5 h in urine was suggestive of malabsorption.Immunoserologically ANA titers were below 1:40, p-ANCA and c-ANCA werenegative, but the IgA anti-Saccharomyces antigen (ASCA) was positive at85 U/mL (normal=<10 U/mL). Based on the increased frequency of waterydiarrhea, abnormal D-xylose absorption, and positive IgA anti-ASCA, thediagnosis of Crohn's disease was made. A therapeutical trial usingcholestyramine was initiated but the frequency of the diarrhea remainedunchanged. In addition, the patient was treated with 230 mg ofmethylprednisolone, and 2×1000 mg of mesalazine. Two years after thistreatment the patient developed entero-enteric fistulae in the terminalileum with sigmoid affection. After admission to the hospital,ileocolectomy was performed and 22 cm of the ileum was resected. Uponhis release remission maintenance with 3×500 mg of mesalazine wasimplemented.

For eight years following this treatment the patient continued to sufferfrom increasing frequency of watery diarrhea and lost an additional 14pounds. During this period several additional treatment attempts weremade using aspirin, loperamide, and budesonide, unfortunately withoutsignificant clinical improvement. Furthermore, the patient was losingmore weight on a monthly basis. A complete review of the medical historyrevealed the fact that almost thirteen years earlier, gliadin IgGantibody had been found to be elevated, which was considered normal atthe time. Since all classical treatments for Crohn's disease had failedto improve the clinical picture over all the years, a comprehensive testfor the assessment of gluten immune reactivity and sensitivity wasordered. This included IgG and IgA against wheat (i.e. mixed wheatantigens), native and deamidated α-gliadin peptides, γ-gliadin,ω-gliadin, glutenin, gluteomorphin, prodynorphin, gliadin-tTg complex,transglutaminase, wheat germ agglutinin, and GAD-65.

Results depicted in Tables 6 and 7, Sample #3 show that the patient hada significant elevation of IgG antibodies against 11 out of 12 testedantigens, and IgA antibodies against mixed wheat antigens, α-gliadin33-mer, ω-gliadin, prodynorphin, wheat germ agglutinin and GAD-65 weredetected at 2-5 fold above the normal range. Based on these results, inaddition to Crohn's disease a diagnosis of gluten sensitivity was alsomade. A diet consisting of rice, potato, and othergluten-free/yeast-free foods was commenced immediately, which led aftersix weeks to a complete cessation of diarrhea. Upon continuation of thegluten-free diet, not only did stool consistency become normal but thepatient also started gaining weight. On follow-up one year later thepatient was back to a normal state and had regained more than 80% of hislost weight.

Discussion: This case reports on the association of Crohn's disease withgluten sensitivity but not with celiac disease. Based on the impressiveclinical response to the gluten-free diet plus the detection of IgG andIgA antibodies against various wheat antigens, and upon re-evaluation ofthe IgG antibody level detected 14 years earlier, the diagnosis ofCrohn's disease with secondary malabsorption and gluten sensitivity wasfinally established. Since IgG antibodies against gliadin but not transglutaminase were detected, it can be argued that in this patient thedisease initiated with gluten sensitivity and not Crohn's disease. Theinitial diagnosis of Crohn's disease was made despite the fact that ademonstration of duration exposure to gluten and risk of autoimmunedisorders was published in 1999 (Ventura A et ai., Gastroenterol, 117:303-310, 1999); unfortunately, this was ignored.

It is contemplated herein that continuous exposure to environmentalfactors such as wheat 15 antigen induced inflammation for a prolongedperiod of time, resulting in inflammatory bowel disease or Crohn'sdisease.

Conclusions Regarding Case Reports: The foregoing case studies show theimportance of proper laboratory testing for confirming diagnoses ofceliac disease, gluten immune reactivity/sensitivity, and autoimmunity.They show that years of erroneous testing and misdiagnoses can lead toyears of suffering. It is vital to get the most accurate information andthe most accurate diagnosis, to distinguish between one condition andanother. FIGS. 5 and 6 show a summary of the current state of testing aswell as a proposed future direction for more accuracy in the diagnosisof celiac disease as proposed by Volta et al. (17). FIG. 7 summarizes aninventive protocol according to certain aspects of the presentinvention, and proposes testing against a repertoire of wheat antigensand peptides so as to provide the most accurate information andconfirmation of celiac disease, Crohn's disease, gluten immunereactivity/sensitivity and autoimmunity.

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the scope of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Where the specification claims refers to at leastone of something selected from the group consisting of A, B, C . . . andN, the text should be interpreted as requiring only one element from thegroup, not A plus N, or B plus N, etc.

REFERENCES

-   1. Catassi C, Fasano A. Celiac disease. Curr Opin Gastroenterol, 24:    687-691, 2008.-   2. Anderson L A, McMillan S A, Watson R G, et al. Malignancy and    mortality in a population-based cohort of patients with coeliac    disease or ‘gluten sensitivity.’ World J Gastroenterol, 13: 146-151,    2007.-   3. Tanabe S. Analysis of food allergen structures and development of    foods for allergic patients. Biosci Biotechnol Biochem, 72: 649-659,    2008.-   4. Chin R L, Latov N, Green P H R, et al. Neurological complications    of celiac disease. J Clin Neuromusc Dis, 5: 129-137, 2004.-   5. Sapone A, Lammers K M, Mazzarella G, et al. Differential mucosal    IL-17 expression in two gliadin-induced disorders: gluten    sensitivity and the autoimmune enteropathy celiac disease. Int Arch    Allergy Immunol. 152: 75-80, 2010.-   6. Sapone A, Lammers K M, Casolaro V, et al. Divergence of gut    permeability and mucosal immune gene expression in two    gluten-associated conditions: celiac disease and gluten sensitivity.    BMC Med. 9: 23, 2011.-   7. Vojdani A, O'Bryan T, Kellermann G H. The immunology of immediate    and delayed hypersensitivity reaction to gluten. Eur. J. Inflamm.    6(1)1-10, 2008.-   8. O, Ram M, et al. Gluten sensitivity in multiple sclerosis:    experimental myth or clinical truth? Ann NY Acad Sci, 1173: 343-349,    2009.-   9. Vojdani A, O'Bryan T, Green J A, et al. Immune response to    dietary proteins, gliadin and cerebellar peptides in children with    autism. Nutr Neurasci. 7(3): 151-161, 2004.-   10. Hadjivassiliou M, Grunewald R A, Lawden M, et al. Headache and    CNS white matter abnormalities associated with gluten sensitivity.    Neural, 56: 385-388, 2001.-   11. Catassi C, et al. Detection of Celiac disease in primary care: a    multicenter case¬20 finding study in North America. Am J    Gastroenterol, 102:1454-1460, 2007.-   12. Sanders D S, et al. Antibody negative coeliac disease presenting    III elderly people-an easily missed diagnosis. BMJ, 330: 775-776,    2005.-   13. Matthias T, Neidhofer S, Pfeiffer S, et al. Novel trends in    celiac disease. Cell. Mol. Immunol. 8: 121-125, 2011.-   14. Jacob S et al. Gluten sensitivity and neuromyelitis optica: Two    case reports. J Neural Neurasurg Psychiatry, 76: 1028-1030, 2005.-   15. Egan C E, et al. Synergy between intraepithelial lymphocytes and    lamina propria T cells drives intestinal inflammation during    infection. Mucosal Immunol, 4: 658-670, 2011.-   16. Kaser A et al. Inflammatory Bowel Disease. Annu Rev Immunol, 28:    573-621, 2010.-   17. Volta U and Villanaci V. Celiac disease: diagnostic criteria in    progress. Cell Mol Immunol, 8: 96-102, 2011.-   18. Vojdani A. 2011. The characterization of the repertoire of wheat    antigens and peptides involved in the humoral immune responses in    patients with gluten sensitivity and Crohn's disease. ISRN Allergy.    Article ID 950104, 1-12.

1-9. (canceled)
 10. A device for characterizing wheat sensitivity in asubject, comprising: a first test region comprising an antigenicα-gliadin peptide; a second test region comprising an antigenicdeamidated α-gliadin peptide; a third test region comprising anantigenic glutenin peptide; a fourth test region comprising an antigenicdeamidated glutenin peptide; and a fifth test region comprising anantigenic complex comprising gliadin and transglutaminase.
 11. Thedevice of claim 10, wherein the antigenic α-gliadin peptide comprises 33or fewer amino acids, and the deamidated α-gliadin peptide comprises 33or fewer amino acids.
 12. The device of claim 11, wherein the antigenicα-gliadin peptide is selected from the group consisting of α-gliadin33-mer of SEQ ID NO. 1 and α-gliadin 17-mer of SEQ ID NO.6, and thedeamidated α-gliadin peptide is selected from the group consisting ofdeamidated α-gliadin 33-mer of SEQ ID NO. 2 and deamidated α-gliadin17-mer of SEQ ID NO.7.
 13. The device of claim 10, wherein the antigenicglutenin peptide comprises 21 or fewer amino acids, and the antigenicdeamidated glutenin peptide comprises 21 or fewer amino acids.
 14. Thedevice of claim 13, wherein the antigenic glutenin peptide comprisesglutenin 21-mer of SEQ ID NO. 15, and the antigenic deamidated gluteninpeptide comprises deamidated glutenin 21-mer of SEQ ID NO.
 16. 15. Thedevice of claim 10, wherein the antigenic complex is selected from thegroup consisting of gliadin-transglutaminase complex A of SEQ ID NO. 19and gliadin-transglutaminase complex B of SEQ ID NO.20.
 16. The deviceof claim 10, further comprising a sixth test region comprising at leastone of an antigenic γ-gliadin peptide and an antigenic ω-gliadinpeptide.
 17. The device of claim 10, further comprising a sixth testregion comprising an antigenic gluteomorphin peptide.
 18. The device ofclaim 10, further comprising a sixth test region comprising an antigenicprodynorphin peptide.
 19. The device of claim 10, further comprising asixth test region comprising an antigenic wheat germ agglutinin peptide.20. A method of characterizing wheat sensitivity in a subject,comprising: identifying the presence of a first antibody of the subjectin a sample from the subject, wherein the first antibody forms a firstcomplex with an antigenic α-gliadin peptide; identifying the presence ofa second antibody of the subject in the sample, wherein the secondantibody forms a second complex with an antigenic deamidated antigenicα-gliadin peptide; identifying the presence of third antibody of thesubject in the sample, wherein the third antibody forms a third complexwith an antigenic glutenin peptide; identifying the presence of a fourthantibody of the subject in the sample, wherein the fourth antibody formsa fourth complex with an antigenic deamidated glutenin peptide; andidentifying the presence of a fifth antibody of the subject in thesample, wherein the fifth antibody forms a fifth complex with anantigenic complex comprising gliadin and transglutaminase.
 21. Themethod of claim 20, wherein the antigenic α-gliadin peptide comprises 33or fewer amino acids, and the deamidated α-gliadin peptide comprises 33or fewer amino acids.
 22. The method of claim 21, wherein the antigenicα-gliadin peptide is selected from the group consisting of α-gliadin33-mer of SEQ ID NO. 1 and α-gliadin 17-mer of SEQ ID NO. 6, and thedeamidated α-gliadin peptide is selected from the group consisting ofdeamidated α-gliadin 33-mer of SEQ ID NO. 2 and deamidated α-gliadin17-mer of SEQ ID NO.
 7. 23. The method of claim 20, wherein theantigenic glutenin peptide comprises 21 or fewer amino acids, and theantigenic deamidated glutenin peptide comprises 21 or fewer amino acids.24. The method of claim 23, wherein the antigenic glutenin peptidecomprises glutenin 21-mer of SEQ ID NO. 15, and the antigenic deamidatedglutenin peptide comprises deamidated glutenin 21-mer of SEQ ID NO. 16.25. The method of claim 20, wherein the antigenic complex is selectedfrom the group consisting of gliadin-transglutaminase complex A of SEQID NO. 19 and gliadin-transglutaminase complex B of SEQ ID NO.20. 26.The method of claim 20, further comprising a sixth test regioncomprising at least one of an antigenic γ-gliadin peptide and anantigenic ω-gliadin peptide.
 27. The method of claim 20, furthercomprising a sixth test region comprising an antigenic gluteomorphinpeptide.
 28. The method of claim 20, further comprising a sixth testregion comprising an antigenic prodynorphin peptide.
 29. The method ofclaim 20, further comprising a sixth test region comprising an antigenicwheat germ agglutinin peptide.